Original Article

Grass from contaminated environments as sources of degrader bacteria and plant growth promoters

M.Cs. Irasema Pérez-Portuondo^{1  *} 

M.Cs. Lidieska Meriño-Reyes² 

Dra.C. Rosa M. Pérez-Silva¹ 

Dra.C. Arelis Abalos-Rodríguez¹ 

Dra.C. Nele Weyens³ 

Dra.C. Ann Cuypers³ 

¹Facultad de Ciencias Naturales y Exactas, Universidad de Oriente. Avenida Patricio Lumumba S/N, CP 90500. Santiago de Cuba. Cuba

²Empresa Geocuba, Santiago de Cuba. Cuba

³Centro de Estudios Ambientales, Universidad de Hasselt, Diepenbeek, Bélgica

ABSTRACT

The search for new strains of bacteria capable of stimulating plant growth or that in bioremediation tasks is essential to the need of developing sustainable agricultural production is used. Plants rhizosphere grown in environments contaminated with organic compounds may be a potential source of compounds bacteria degrade and with characteristics of plant growth promoters. This work aimed to isolate bacteria with characteristics of plant growth promoters from the rhizosphere of plants grown on contaminated soils with petroleum and phenolic compounds. Also the ability to grow using 2,4-dichlorophenoxyacetic acid, 2,4-dichlorophenol and 4-chlorophenol as the sole carbon source was evaluated. Fifty-one bacteria from rhizosphere grasses of Cyperus rotundus, Cynodon dactylon and Scleria sp were isolated. At presence of 2,4-dichlorophenoxyacetic acid it grew 92 %, and in 2,4-dichlorophenol and 4-chlorophenol 45 % grew. The 49 % of the isolates showed two or more characteristics of plant growth promoters of which 16 % also grew in phenolic compounds. These isolates were selected to evaluate degradation of 2,4-dichlorophenol and showed between 15 - 40 % degradation. In conclusion, it is the first reports of rhizobacteria isolated from these weeds with capacity to degrade phenolic compounds. Results suggest the potential of these grasses to isolate bacteria with ability to promote plant grow as well as to degrade chlorophenols and the possibility of their associating to plants for phytoremediation.

Key words: rhizobacteria; biodegradation; chlorophenols; Cynodon dactylon; Cyperus rotundus

INTRODUCTION

Undesirable plants, among which many herbaceous species are considered, provide multiple benefits because, among others, they perform vital services for ecosystems such as the protection and restoration of exposed or degraded soils ¹. Despite all the advantages, they offer for humans and agriculture, the greatest attention remains directed towards their negative impacts. In agriculture, they are a cause for concern because they compete with crops for growth factors such as water, light, nutrients, space and perhaps under certain conditions, carbon dioxide (essential for photosynthesis). The interference of these plants affects agricultural yields and increases the costs of agrotechnical operations and crop processing ².

For this reason, agricultural practices are directly related to the use of herbicides and other pesticides, in order to control pests that attack crops and thus increase food productivity. However, the environmental pollution caused by these pesticides constitutes a problem of great importance on a global scale, due to the different levels of persistence of these compounds in the environment, as well as the toxicity exerted on domestic animals, birds, fish, insects and other representatives of wildlife ³ .

Among the herbicides more used are phenoxy-herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) or e l 4-chloro-2-methylphenoxyacetic (MCPA). The degradation of these compounds leads to the formation of phenols (phenol, 2-chlorophenol and 2,4-dichlorophenol) and catechols (catechol and 4,6-dichlorocatechol). The biotransformation of 4-chlorophenoxyacetic acid leads to the formation of 4-chlorophenol and photodegradation of Declorprop (2,4-dichlorophenoxy-2-propionic acid) and 2,4-D cause the formation of 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol. These compounds are dangerous by the regulations of environmental protection agencies ⁴.

On the other hand, agriculture also demands the use of fertilizers, as well as growth stimulants, to increase agricultural production under conditions, every day more accentuated, with low soil fertility and abiotic stressors. Among the latter, drought, salinity, and air and soil pollution stand out due to the presence of heavy metals and pesticides, which act as the main limiting factors in crop production, because they affect all plant functions.

The use of plant growth promoting bacteria to reduce the use of chemical compounds in agriculture is becoming increasingly important in the international context as a way to guarantee environmental and food quality. Currently, multiple efforts are being made for the commercial exploitation of these bacteria as bioinoculants for various crops of economic importance ^5,6 .

At the same time, the use of microorganisms to eliminate xenobiotics and persistent organic compounds is an ecosystem friendly process. Microbial activity near the roots (rhizosphere) offers a favorable environment for the cometabolism of recalcitrant chemical compounds, many of which, due to their nature, cannot be taken directly by plants. Several researchers has reported that bacteria that live in the rhizosphere of plants that grow in contaminated land are more likely to degrade toxic compounds, constituting a potential source of bacteria tolerant to the compounds present in that environment and with the ability to metabolize them ^(7.8).

With the aim of obtaining bacteria that are promoters of plant growth and degraders of xenobiotic compounds, the rhizosphere of many crop plants has been studied ⁹; however, undesirable herbaceous plants have long been ignored ¹⁰. Increasingly the investigations where the plants are scanned herbaceous as sources of plant growth promoting rhizobacteria, due to the wide biodiversity that can display them ⁶. Many of its species are capable of growing in contaminated environments if it is added ^11-13, then these could also be used for the isolation of bacteria that meet both capacities.

This work has as objective isolating rhizospheric bacteria from herbaceous plants grown in sites contaminated with phenolic compounds, capable of exhibiting characteristics promoting plant growth and using phenolic compounds as the sole source of carbon.

MATERIALS AND METHODS

Insulation of rizobacteria from plants living in contaminated soils

Specimens were collected three plant species growing in the area of movement and storage of products (MAP) in Refinery “Hermanos Díazˮ, Santiago de Cuba province. This is an area of 56 m ² , contaminated with hydrocarbons and phenolic compounds characterized by the presence of a sandy soil (76.7 % sand, 6.9 % gravel and 16.4 % clay), with pH 7, 2 and 24.7 % field capacity, whose composition in phosphate and nitrate is 5.60 x 10 ^-2 and 3.82 mg g ^-1 of soil , respectively (14) . Samples of the herbaceous plants as Cyperus rotundus L, Scleria sp. and Cynodon dactylon (L), were taken.

In the work, plants were preserved with the land around them and taking all the roots carefully, without this involvement and deposited in plastic bags for transport.

Rhizobacteria are isolated rum according to the methodology described by Woyessa and Assefa ¹⁵. For this, soil was removed carefully from the roots and then these were washed with sterile distilled water to remove as many bacteria as possible. 10 g of the rhizosphere soil of each plant was mixed by stirring with 90 mL of the washing water from the roots of the plant itself. Serial dilutions were made from each suspension obtained from each plant, using MgSO ₄ 7H ₂ O 0.01 mol L^-1. Subsequently, 0,1 mL of each suspension, as well as the corresponding dilutions, was seeded by spread plating with nutrient agar. Plates at 37 °C for 48 h were incubated. Representative colonies were selected of all morphological types, which were subcultured on nutrient agar medium for purification. The isolates obtained are purified by successive replantings on nutrient agar plates and was preserved in wedges of the same medium at 4 °C .

Evaluation of the promoter characteristic presence of vegetable growth in the isolated bacteria

The evaluation was carried out based on the presence or absence of the following characteristics:

• Production of organic acids acetoin and indoleacetic acid

• Fixation of atmospheric nitrogen

• Solubilization of calcium phosphate.

The production of organic acids was evaluated by the colorimetric method of Cunningham and Kuiack ¹⁶, adding the indicator to 0.1 % red lizarin to bacterial cultures grown for 5 days in sucrose-tryptone (ST) medium. Changing color, from pink to yellow that indicated response positive.

Acetoin production was detected by inoculating the bacteria in Voges-Proskauer medium. After 48 hours of incubation a colorimetric response was induced according to Romick and Fleming ¹⁷, taking as a positive response the appearance of a reddish-pink color.

The test to detect the bacteria that solubilize the phosphate was developed according to Nautiyal ¹⁸. The presence of a clear halo around the colonies indicates phosphate-solubilizing capacity.

The Salkowsky test (adapted from Patten and Glick) ¹⁹ was used to evaluate the ability to produce indoleacetic acid (AIA). The bacteria were inoculated in 1 mL of 869 1/10 medium supplemented with 0.5 g L^-1 of tryptophan. After incubating for 4 days a colorimetric reaction was induced by adding the Salkowsky reagent (Gordon and Weber) ²⁰. The appearance of a pink color was taken as positivity.

The ability to fix nitrogen was evaluated by the method described by Xie et al²¹. The isolated bacteria were grown in the N-medium supplemented with blue bromothymol (with and without NH ₄ Cl as a nitrogen source) and incubated for two weeks at 30 ° C. El color change from blue to yellow was taken as a response positive to the test .

All trials were performed in triplicate.

Growth in the mining environment with chlorophenol as the only carbon source

The following analytical grade chlorophenolic compounds were used: 4-chlorophenol (Merck), 2,4-dichlorophenol (SIGMA-ALDRICH), 2,4-dichlorophenoxyacetic acid (2,4-D) (SIGMA-ALDRICH).

To select bacterial isolates capable of growing in the presence of chlorophenols as the only carbon source, a chlorine-free mineral medium (MMS) was used. It consists in 0.02 mol L^-1 phosphate buffer (KH₂O₄ , Na₂HPO₄ , pH 7.2), 0.5 g L^-1 of (NH₄)₂ SO₄ and 0.2 g L ^-1 of MgSO_4, 7H₂ O in distilled water, supplemented with 10 mL of a solution of elements trace comp et up of (in mg L^-1): Ca (NO₃ )_2, 4H₂ O, 600; FeSO₄ .7H ₂ O, 200; MnSO₄ . 4H₂ O, 20; CuSO_4, 5H₂ O, 40; ZnSO₄. 7H₂ O, 20; H₃BO₃ 3; NaMoO₄ . 2H₂ O, 4 and acidified with 1 mL of concentrated sulfuric acid L ^-1.

To medium was added agar (1.5 g L ^-1 ) , bromothymol blue indicator (0.04 g L ^-1 ) , and the herbicide chlorophenoxy 2,4- D (0.2 g L ^-1 ). The isolates that were able to grow and produce color changes in the colony or in the medium from blue to yellow were declared positive to the degradation test ²².

The isolates that were positive to assay sembra rum then on plates containing the same medium, but substituting 2,4-D by 4-chlorophenol or 2,4-dichlorophenol as unique s source s carbon , both at a concentration 0.02 g L ^-1 . The plates s and incubated rum at 30 ° C for 5 days to assess growth. Media plates that were not inoculated were used as controls. The tests were performed in duplicate.

Bacteria that were able to grow in the presence of both chlorophenols were considered capable of degrading the phenolic compounds tested ⁹ and rum was selected for subsequent studies.

Evaluation tests of the degrading capacity of selected rizosphere 2,4-diclorophenol isolated

Preparation of inoculum bacterial

The selected bacterial isolates were inoculated in 10 mL of nutrient broth. They were incubated for 72 h at 33 °C with stirring. The biomass was separated by the centrifugation and it was washed twice with MgSO, 7H ₂ O 0.01 mol L^-1.

Biodegradation of 2,4-dichlorophenol

Biodegradation was carried out in distilled water using 5 % wet biomass, 0.2 g L^-1 of chlorophenol and a reaction volume of 2 mL. The reaction was run under stirring at 30 °C, for 24 h. Is LLEV or parallel control assay without biomass ²³. To the supernatant, the analyzes of phenols and chloride were performed. The quantification of chlorophenol is performed by means of the colorimetric method of 4-aminoantipyrine , according modifying Farrell and Quilty ²⁴ the procedure described in Standard Methods for the Determination of Water and Wastewater ²⁵, while the chloride concentration was analyzed colorimetrically according to Jörg and Bertau ²⁶. The degradation percentage of chlorophenols was determined according to equation.

$\% Degradation=\left(\frac{C_i-C_f}{C_i} \right)*100$

Where C_i was the initial concentration of the compound and C_f the final concentration. The chloride was quantified by the equation of the straight line of the sodium chloride standard curve (0.00 3- 0.1 g L^-1).

Statistical analysis

Statistical analysis of experimental data was performed using the STATGRAPHICS Centurion XV program. Simple classification analysis (ANOVA I) and multiple range test was performed based on the significant difference procedure minimum (LSD) of Fisher for comparison of means. In all cases, 5 % significance was used. These tests were used to compare the degradation chlorophenol and release of chloride between isolates.

RESULTS AND DISCUSSION

The collection of plants was performed in a localized area in the gasoil refinery characterized by soil contamination with oil and phenolic compounds derived from the refining process.

In the field a lot of herbaceous plants and some bushy specimens were observed. The herbaceous collected belonged to Cyperaceae family: Cyperus rotundus L and Scleria sp and to the Poaceae Family: Cynodon dactylon (L), all from Monocotyledonea class.

C. dactylon (bermuda grass or common grass) and C. rotundus (corocillo or coquito) are two herbaceous that are considered as invasive pests of many crops, with allelopathic effects due to the release to the soil of multiple allelochemical compounds that affect the development of the neighboring plants ^27,28.

These plants have been referenced by some authors as species that grow in soils contaminated with hydrocarbons and that can degrade oil and diesel ^29,30 . Sarathambal and Llamurugu used them as a source of nitrogen fixing rhizobacteria and phosphate solubilizers ⁵.

Other authors ^31,32 described C. dactylon as useful for the rehabilitation of soils exploited by mining and soils contaminated with heavy metals.

The followed procedure of rizobacteria allowed the isolation of 51 bacteria, being the highest number of herbaceous plants isolated in C. rotundus and Scleria sp. (Table 1).

Table 1 Distribution of rhizobacteria according to the source of isolation 

Plants	Amount of bacteria isolated
C. rotundus	20
Scleria sp	17
C. dactylon	14
Total	51

Characteristics of promoters of vegetable growth of isolated rhizobacteria

Table 2 shows a summary of rhizobacteria isolated from each plant had more than one property of promoting plant growth is shown. As it can observed, the appearance of these characteristics was markedly influenced the origin of bacteria, that is, the plant from which they came. This finding suggests that the plant species influences the differential colonization of the rhizosphere by certain bacterial groups, even when these plant species grow in the same environment

Table 2 Characteristics of plant growth promoters in rhizobacteria isolated from plants grown in soils contaminated with hydrocarbons 

	Isolated	Acetoin Production	Organic acid production	Phosphate Solubilization	Fixation of nitrogen	Production of A I A
Cyperus rotundus	RR-42	-	+	+	-	+
	RR-45	-	+	+	-	-
	RR-47	+	-	+	-	-
	RR48	-	+	+	-	-
	RR-50	-	+	+	-	-
	RR-51	-	+	+	-	-
	RR-52	-	+	+	-	-
	RR-53	-	+	+	-	-
	RR-54	-	+	+	-	-
	RR-56	-	+	+	-	+
	RR-102	-	+	+	-	+
Scleria sp	RR-62	-	+	+	-	-
	RR-63	-	+	+	-	-
	RR-64	-	+	+	-	-
	RR-69	-	+	+	-	-
	RR-72	-	+	+	-	+
	RR-75	-	+	+	-	-
Cynodon dactylon	RR-76	-	+	+	-	-
	RR-79	-	+	+	-	+
	RR-81	-	+	+	+	+
	RR-84	-	+	+	+	+
	RR-85	-	+	+	-	+
	RR-86	+	+	-	-	-
	RR-87	+	+	-	-	-
	RR-88	+	+	-	-	-

To compare, for each plant species, the percentage of isolates that showed characteristics of growth promoters, it was found that C. rotundus contributed 5 % of isolates capable of producing acetoin and indoleacetic acid, 65 % with ability to produce organic acids and 60 % phosphate solubilizers. In Scleria sp. 71 % of isolates producing organic acids and 53 % of phosphate solubilizers were observed, as well as 6 % of producers of indolacetic acid; no nitrogen fixers or acetoin producers were isolated. It was observed that 36 % of isolates obtained from C. dactylon solubilized phosphates ; however , the 100 % produced organic acids, while four isolates produced indoleacetic acid and two were able of fixing atmospheric nitrogen, thus being same l to plant more producers provided isolated acetoin (21 %).

Other authors hypothesized that the interactions between plants and soil microorganisms are highly dynamic in nature and are based on co-evolutionary pressures, so it is not surprising to find that the microbial community in the rhizosphere differs between species including genotypes within species, and even between different stages of plant development ³³.

It was found that the ability to fix nitrogen and produce acetoin were the least frequent, while the predominant characteristic was to produce organic acids, obtaining between three and four isolates with the first characteristics and 39 bacteria producing organic acids.

Several authors have argued that organic acids produced by phosphate solubilizing bacteria are not determinants in solubilization. The only determining factor is the concentration of hydronium ions (H₃ O⁺), those that occur in respiration or as a consequence of assimilation of ammonium ions (NH₄ ⁺), which alters the pH of medium, enough as to mobilize minerals in the soil ^34,35.

In this work, the capacities of producing organic acids and solubilizing the insoluble inorganic phosphorus were evaluated finding that both characteristics were independent in some of the isolated bacteria, which suggests the existence of several solubilization mechanisms.

Nahas supported that there were several mechanisms, which included not only the excretion of organic acids, but also the extrusion of protons or the production of chelating agents ³⁶. Other mechanisms such as, the production of inorganic acids such as sulfuric acid, nitric acid or carbonic acid have been mentioned by other authors ³⁷.

The literature reviewed collects data on the isolation of bacteria that promote plant growth of rhizosphere of plants that are valuable for the economy, such as cotton, wheat, corn ³⁸ and rice ³⁹. Despite this, more and more works are being explored where other herbaceous plants are explored as a source of rhizobacteria with promoter characteristics, due to the wide bacterial diversity they can show ^5,10 . This research becomes another contribution to the use of these plants as sources of plant growth promoting rhizobacteria. These new isolates, carriers of these valuable qualities, could be used soils with deficient nutriments to mitigate stress and achieve sustainability of crops ⁵.

The plants undesirable can be harmful for crops, because many of them produce toxic substances that inhibit crop growth (allelopathy) or compete with these by light, space and nutrients. On the other hand, when they grow where they do not affect crops or during rotation of these it can take advantage of many of the beneficial properties they offer. These are protection of soil erosion, restoration of biodiversity, absorption, preservation and recycling of soluble nutrients, which would be filtered otherwise through the soil, as well as the restoration of exposed or degraded soils ¹. Some plants herbaceous tolerated compounds and toxic elements even degrade or build-up and an inside, which is another aspect of great value for these plants, aspect corroborated by isolating plants contaminated with hydrocarbons and phenolic environments.

Due to the use of chemicals such as pesticides often, it remains a residual concentration or is more toxic intermediates produced during degradation, so that contamination is generated on the sites where these compounds are used. Some pesticides with heavy metals in their structure (copper hexachloride), which tend to accumulate in the soil or in plants. This contamination may spread to surface waters and groundwater, transferring contamination to other more sensitive ecosystems or are trapped in the particles of soil it difficult to remove. These pollutants can remain in ecosystems even when their use has been abandoned, being able to accumulate in living organisms and concentrate their toxic effect on the food chain causing damage not only to white species but also to other beneficial species such as bees, fish, microorganisms from the ground, as well as man ⁴⁰.

All of the foregoing supports the enormous perspectives offered by these plants for possible bioremediation strategies of agricultural soils, contributing not only as a reservoir of plant growth stimulating bacteria, but also for their own implications in contaminant removal processes.

Capacity of the rizobacteria of growing in mineral environment with chlorophenols as the only carbon source

To determine the ability of the bacteria rhizosphere isolates grow in different chlorophenoxy compounds and selecting the best behavior in all compounds tested, one was performed growth assay using 2,4-D acid as the sole source of carbon and energy (Table 3). From 51 isolates tested 9 2 % showed capacity to grow on 2,4-D, suggesting that this compound can degrade to use as carbon source for biosynthesis cell material.

Table 3 Growth in chlorophenols of isolated rhizobacteria 

	2,4-D (0.2g L^-1)	2,4-DCF (0,02g L^-1)	4-CF (0.02g L^-1)	2,4-DCF and 4-CF (0,02g L^-1)
C.rotundus	17 ( 85)	8 (40)	5 (25)	3 ( 15)
Scleria sp	16 ( 94)	14 (82)	12 (71)	11 ( 65)
C. dactylon	14 ( 100)	10 (71)	10 (71)	9 ( 64)
Total	4 7 (92)	32 ( 63)	27 ( 53)	23 ( 45)

In brackets are the percentages with respect to the total in each plant

Plant roots exude many phenolic compounds. Microorganisms capable of using these phenolic compounds as a carbon source often have enzymes that can co-metabolize contaminants with similar structure ⁴¹. Many of these compounds, such as naringin, myricetin, catechin and others are released by plant roots and have been shown to stimulate the growth of polychlorinated biphenyls (PCB) degrading bacteria. Some authors have shown that terpene exudate (eg α-pinene, α-terpinen, among others) can act on the activity of the enzyme biphenyl dioxygenase in PCB degrading bacteria, activating or inhibiting it ⁴².

The literature suggests that there are two routes by which 2,4-D can be degraded. The first route involves the alpha-ketoglutarate dioxygenase to give rise to 2,4-dichlorophenol, while the other route begins by activating a dehalogenase to form 4-chlorophenoxyacetate, which transforms into 4-chlorophenol ⁴³. This knowledge indicates the need to explore whether bacteria that are capable of growing in 2,4-D are capable of using either or both of the two main intermediates of these routes: 2,4-dichlorophenol and 4- chlorophenol

The discovery of bacteria that can grow in both compounds suggests that these bacteria can degrade any of them without being inhibitory for growth (Table 3). This result means that these bacteria can be used to remedy soils that could be contaminated even with both compounds.

The number and type of bacteria found in different soil may be influenced by Asian or by its conditions, including temperature, humidity and the presence of inorganic and organic compounds (salts) as well as by the number and type of plant in they grow.

The results showed that Scleria sp and C. dactylon were the ones that contributed the most in rhizosphere isolates with the ability to use chlorophenols. The finding coincides with that raised by other authors ⁴⁴, by offering evidence that plant species prepare the bacterial soil community and that their exudates select the bacteria that live in their roots from the soil community in its whole.

According to the results obtained, those bacteria that were able to grow in the tested chlorophenols were chosen and, in addition, they had several characteristics that promoted plant growth, with eight isolates being selected.

Bacteria capable of meeting both characteristics sought (growth in the presence of pesticides and properties of plant growth promoters) have been described by several authors ^35,45, the most mentioned compounds being 2,4-D, pentachlorophenol, trichlorophenol and 4 -chlorophenol, but no references have been found so far on the degradation of 2,4-dichlorophenol by rhizobacteria. However, Ahemad and Saghir referred to the tolerance of rhizobacteria isolated by them to fungicides and herbicides whose chemical structure was 4-chlorophenol and 2,4-dichlorophenol ⁹.

Degradation of chlorophenolic compounds by selected rizobacteria

When evaluating the degradation of 2,4-DCF (0.2 g L^-1 ), the main intermediary of the degradation of the herbicide 2,4-D, by selected rhizobacteria, the suggested degrading capacity when growing in this compound as unique was corroborated carbon source (Table 4). The results achieved showed that the RR72 isolated is the one that presented the best features. The appearance of free chloride confirmed the degradation of chlorophenol.

Table 4 Examination of the isolates for the degradation of 2,4-dichlorophenol 

Isolated	Biodegradation (%)
RR56	22 a
RR62	22 a
RR72	40 b
RR79	18 c
RR81	22 a
RR84	15 d
RR88	19 c
RR102	6 e

For p <0.05, different letters show significant differences

Other authors found a degradation of 15 % at 24 h and between 70-85 % at 7 days when they tested this compound at the same concentration (0.2 g L ^-1 ) ⁴⁶. It was attributing to the toxicity of the compound and the dose used the behavior of the bacteria because they reported up to 98 % degradation for concentrations less than 0.1 g L^-1. The results showed that e l procedure used was able to assess in a rapid manner capable of degrading microorganisms of interest, allowing or using as a method of screening led to the selection of bacteria with high chlorophenol degradability.

To date, there are reports of plant growth promoting rhizobacteria isolated from species of Scleria, so that the results would be the first communication on this finding. Similarly, there is no background of rhizobacteria isolated Scleria, C. rotundus and C. dactylon grow using 4-chlorophenol, 2,4-dichlorophenol and 2,4-D as the only source of carbon.

CONCLUSIONS

• 51 isolated from the rhizosphere were obtained of three herbaceous plants grown in contaminated soils phenolic compounds and hydrocarbons, which demonstrates the practical utility of these plants as a source of bacteria with characteristics promising for bioremediation applications.

• The herbaceous C. dactylon provided the greatest amount of isolated producers of acetoin and organic acids and bacteria fixing atmospheric nitrogen, desired to formulate the characteristics biofertilizer.

• 92 % of the isolated bacterial grew in the presence of the herbicide 2,4-D as the only carbon source which suggest their ability to degrade.

• Herbaceous Scleria sp. and C. dactylon provided the highest proportion of rhizospheric isolates capable of using the intermediate chlorophenols of the degradation of 2,4-D (2,4-DCF and 4-CF) as the only carbon source .

• Of the eight bacteria selected, seven showed the ability to degrade 2,4-DCF, the main intermediary of the degradation of 2,4-D and raw material to obtain it.

RECOMMENDATIONS

• The results achieved in this work recommend evaluating the capacity of the isolates obtained as plant growth stimulators. Also, use these isolates to mitigate crop stress caused by nutrient deficiency, as well as in the substitution of chemical fertilizers.

• To assess the capabilities of these isolates for decontamination of soils treated with the 2,4-D herbicide or other phenolic- based pesticides .

ACKNOWLEDGEMENT

The authors are grateful for the funding provided by the P1 project “Environmental Scientific Services for the development of sustainable agriculture and confronting climate change in eastern Cuba”, within the VLIR-IUC project established between the Flemish Universities of Belgium with the University of East.

REFERENCES

1. Schonbeck M. An ecological understanding of weeds. Revista Extensión. 2013;20:1-7.

2. Qasem JR, Foy CL. Weed allelopathy, its ecological impacts and future prospects: a review. Journal of Crop Production. 2001;4(2):43-119. doi:10.1300/J144v04n02_02

3. Verma JP, Jaiswal DK, Sagar R. Pesticide relevance and their microbial degradation: a-state-of-art. Reviews in Environmental Science and Bio/Technology. 2014;13(4):429-66. doi:10.1007/s11157-014-9341-7

4. Kotresha M. Degradation of phenol by novel strain Pseudomonas aeruginosa MTCC 4997 isolated from petrochemical industrial effluent. International Journal of Microbial Resource Technology. 2014;2(3):7-15.

5. Sarathambal C, Ilamurugu K. Phosphate solubilising diazotrophic bacteria associated with rhizosphere of weedy grasses. Indian Journal of Weed Science. 2014;46(4):364-9.

6. Ashok K, Bihari RM, Richa R. Characterization of bacterial strains and their impact on plant growth promotion and yield of wheat and microbial populations of soil. African Journal of Agricultural Research. 2015;10(12):1367-75. doi:10.5897/AJAR2014.8894

7. Balseiro-Romero M, Gkorezis P, Kidd PS, Vangronsveld J, Monterroso C. Enhanced degradation of diesel in the rhizosphere of after inoculation with diesel-degrading and plant growth-promoting bacterial strains. Journal of Environment Quality. 2016;45(3):924. doi:10.2134/jeq2015.09.0465

8. Kala DS. Rhizoremediation: a promising rhizosphere technology. IOSR Journal of Environmental Science, Toxicology and Food Technology. 2014;8(8):23-7. doi:10.5772/56905

9. Ahemad M, Khan MS. Assessment of pesticide-tolerance and functional diversity of bacterial strains isolated from rhizospheres of different crops. Insight Microbiology. 2011;1(1):8-19. doi:10.5567/IMICRO-IK.2011.8.19

10. Sarathambal C, Ilamurugu K, Priya LS, Barman KK. A review on weeds as source of novel plant growth promoting microbes for crop improvement. Journal of Applied and Natural Science. 2014;6(2):880-6.

11. Khoramnejadian S, Khoramnejadian S. Phytoremediation of heavy metals and total petroleum hydrocarbons by amaranth. Journal of Biology and Today's. World. 2013;2(12):514-6.

12. Tsuboi S, Yamamura S, Nakajima-Kambe T, Iwasaki K. Diversity of alkane hydroxylase genes on the rhizoplane of grasses planted in petroleum-contaminated soils. Springer Plus. 2015;4(1):514-6. doi:10.1186/s40064-015-1312-0

13. Mishra T, Pandey VC, Singh P, Singh NB, Singh N. Assessment of phytoremediation potential of native grass species growing on red mud deposits. Journal of Geochemical Exploration. 2017;182:206-9. doi:10.1016/j.gexplo.2016.12.015

14. Nápoles J, Rodríguez S, Santiago L, Ábalos A. Disminución del extracto orgánico total en suelos contaminados con hidrocarburos. Tecnología Química. 2015;35(3):322-33.

15. Woyessa D, Assefa F. Diversity and plant growth promoting proerties of rhizobacteria isolated from tef (Eragrostis tef). Ethiopian Journal of Education and Sciences. 2011;6(2):81-94.

16. Cunningham JE, Kuiack C. Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii. Applied and Environmental Microbiology. 1992;58(5):1451-8.

17. Romick TL, Fleming HP. Acetoin production as an indicator of growth and metabolic inhibition of Listeria monocytogenes. Journal of Applied Microbiology. 1998;84(1):18-24.

18. Nautiyal CS. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology Letters. 1999;170(1):265-70. doi:10.1111/j.1574-6968.1999.tb13383.x

19. Patten CL, Glick BR. Role of Pseudomonas putida indoleacetic acid in development of the host plant root system. Applied and Environmental Microbiology. 2002;68(8):3795-801.

20. Gordon SA, Weber RP. Colorimetric estimation of indoleacetic acid. Plant Physiology. 1951;26(1):192-5.

21. Xie GH, Cui Z, Yu J, Yan J, Hai W, Steinberger Y. Identification ofnif genes in N2-fixing bacterial strains isolated from rice fields along the Yangtze River Plain. Journal of Basic Microbiology. 2006;46(1):56-63. doi:10.1002/jobm.200510513

22. Kröckel L, Focht DD. Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event. Applied and Environmental Microbiology. 1987;53(10):2470-5.

23. Bhaskar G, Rita V, Rita V, Rita V. Biodegradation of chlorobenzene and chlorophenols by Pseudomonas cultures. Research Journal of Chemistry and Environment. 2013;17(7):40-3.

24. Farrell A, Quilty B. Degradation of mono-chlorophenols by a mixed microbial community via a meta- cleavage pathway. Biodegradation. 1999;10(5):353-62. doi:10.1023/A:1008323811433

25. Rice EW, Baird RB, Eaton AD. Standard methods for the examination of water and wastewater ENT#091;InternetENT#093;. 23rd Edition. American Public Health Association, American Water Works Association, Water Environment Federation; 2017 ENT#091;cited 2019 Apr 8ENT#093;. 1350 p. Available from: https://store.awwa.org/store/productdetail.aspx?productId=65266295

26. Jörg G, Bertau M. Thiol-tolerant assay for quantitative colorimetric determination of chloride released from whole-cell biodehalogenations. Analytical Biochemistry. 2004;328(1):22-8. doi:10.1016/j.ab.2004.01.027

27. Geethambigai, CS, Prabhakaran, J. Allelopathic influence of Cyperus rotundus L. and Cynodan dactylon L. on physico-chemicaland biological properties of soil. International Journal of Current Biotechnology. 2014;2(11):5-8.

28. Ameena M, Geethakumari VL, Sansamma G. Allelopathic influence of purple nutsedge (Cyperus rotundus L.) root exudates on germination and growth of importantfield crops. International Journal of Agricultural Sciences. 2014;10(1):186-9.

29. Basumatary B, Saikia R, Bordoloi S. Phytoremediation of crude oil contaminated soil using nut grass, Cyperus rotundus. Journal of Environmental Biology. 2012;33(5):891-6.

30. Subhashini V, Swamy AVVS. Phytoremediation of cadmium and chromium contaminated soils by Cyperus rotundus. American International Journal of Research in Science, Technology, Engineering & Mathematics. 2014;6(1):97-101.

31. Singh K, Pandey VC, Singh RP. Cynodon dactylon: An efficient perennial grass to revegetate sodic lands. Ecological Engineering. 2013;54:32-8. doi:10.1016/j.ecoleng.2013.01.007

32. Srinivasan D, Ilavenil KK. Bioremediation of dyes and heavy metal ions using Cynodon dactylon - A Review. Journal of Chemical and Pharmaceutical Sciences. 2017;10(1):363-70.

33. Keshav PS, Shivesh S, Kumar S, Vasudha S, Kirti T, Sphoorti S. Nature and role of root exudates: Efficacy in bioremediation. African Journal of Biotechnology. 2013;10(48):9717-24. doi:10.5897/AJB10.2552

34. Souza R de, Ambrosini A, Passaglia LMP. Plant growth-promoting bacteria as inoculants in agricultural soils. Genetics and Molecular Biology. 2015;38(4):401-19. doi:10.1590/S1415-475738420150053

35. Rajasankar R, Manju Gayathry G, Sathiavelu A, Ramalingam C, Saravanan VS. Pesticide tolerant and phosphorus solubilizing Pseudomonas sp. strain SGRAJ09 isolated from pesticides treated Achillea clavennae rhizosphere soil. Ecotoxicology. 2013;22(4):707-17. doi:10.1007/s10646-013-1062-0

36. Nahas E. Factors determining rock phosphate solubilization by microorganisms isolated from soil. World Journal of Microbiology & Biotechnology. 1996;12(6):567-72. doi:10.1007/BF00327716

37. Seshadri S, Muthukumarasamy R, Lakshminarasimha C, Ignacimuthu S. Solubilization of inorganic phosphates by Azospirillum halopraeferans. Current Science. 2000;79(5).

38. Mohite B. Isolation and characterization of indole acetic acid (IAA) producing bacteria from rhizospheric soil and its effect on plant growth. Journal of Soil Science and Plant Nutrition. 2013;13(3):638-49. doi:10.4067/S0718-95162013005000051

39. Tan KZ, Radziah O, Halimi MS, Khairuddin AR, Habib SH, Shamsuddin ZH. Isolation and characterization of rhizobia and plant growth-promoting rhizobacteria and their effects on growth of rice seedlings. American Journal of Agricultural and Biological Sciences. 2014;9(3):342-60. doi:10.3844/ajabssp.2014.342.360

40. Chaudhry Q, Schröder P, Werck-Reichhart D, Grajek W, Marecik R. Prospects and limitations of phytoremediation for the removal of persistent pesticides in the environment. Environmental Science and Pollution Research. 2002;9(1):4-17. doi:10.1007/BF02987313

41. Musilova L, Ridl J, Polivkova M, Macek T, Uhlik O. Effects of secondary plant metabolites on microbial populations: changes in community structure and metabolic activity in contaminated environments. International Journal of Molecular Sciences. 2016;17(8):1205. doi:10.3390/ijms17081205

42. Oh ET, Koh SC, Kim E, Ahn YH, So JS. Plant terpenes enhance survivability of polychlorinated biphenyl (PCB) degrading Pseudomonas pseudoalcaligenes KF707 labeled with gfp in microcosms contaminated with PCB. Journal of Microbiology and Biotechnology. 2003;13(3):463-8.

43. Arora P, Bae H. Bacterial degradation of chlorophenols and their derivatives. Microbial Cell Factories. 2014;13(1):31. doi:10.1186/1475-2859-13-31

44. Huang X-F, Chaparro JM, Reardon KF, Zhang R, Shen Q, Vivanco JM. Rhizosphere interactions: root exudates, microbes, and microbial communities. Botany. 2014;92(4):267-75. doi:10.1139/cjb-2013-0225

45. Shaheen S, Sundari K. Exploring the applicability of pgpr to remediate residual organophosphate and carbamate pesticides used in agriculture fields. International Journal of Agriculture and Food Science Technology. 2013;4(10):947-54.

46. Patel BP, Kumar A. Optimization study for maximizing 2,4-dichlorophenol degradation by strain using response surface methodology and kinetic study. Desalination and Water Treatment. 2015;57(39):18314-25. doi:10.1080/19443994.2015.1091988

Received: March 26, 2018; Accepted: March 19, 2019

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